P. aeruginosa is a frequent cause of severe acute and chronic lung infections in humans, but the innate immune response to this organism is not yet fully understood. Published data indicate the MyD88 signaling pathway is critical in this response, yet toll-like receptors (TLRs) 2 and 4, which signal through MyD88, are not essential for bacterial clearance. TLR5 also signals through MyD88 and is necessary for the response to purified flagellin in the lung. When exposed to very high doses of intranasal P. aeruginosa, mice lacking TLR5 have a similar mortality rate as wild type mice, but those lacking both TLRs 4 and 5 have decreased survival. This proposal has two specific aims: 1. Characterize in greater mechanistic detail the roles of TLR4 and TLR5 in the innate immune response to acute P. aeruginosa lung infection by exposing wild-type, TLR4-/-, TLR5-/- and TLR4/5-/- mice to aerosolized wild type and nonflagellated P. aeruginosa. Lung inflamm of P. aeruginosa. However, these proteins have a synergistic relationship; when both are absent, inflammatory response and bacterial clearance will be significantly impaired. 2. Determine the cell specificity of the TLR4 and TLR5 responses to P. aeruginosa by culturing primary alveolar macrophages and airway epithelial cells from wild-type and knockout animals, followed by stimulation with P. aeruginosa and subsequent measurements of inflammatory response. Complementary experiments will use inhibitory RNA to create knockdown models of airway epithelial and alveolar macrophage cell lines to confirm the findings in primary cells. Finally, immunohistochemical techniques will be used to study the timing of NF-kB translocation to the nuclei of epithelial cells after in vivo infection. The current paradigm holds that flagellin-TLR5 signaling is the primary method of epithelial cell stimulation when exposed to P. aeruginosa, whereas the LPS-TLR4 axis drives the alveolar macrophage response. The truth is likely more complicated, with both TLRs playing a larger role than previously supposed in both cell types. [unreadable] PUBLIC HEALTH RELEVANCE: P. aeruginosa is a clinically important bacteria in human lung disease. This is a proposal to study proteins in the lung that trigger the immune response to P. aeruginosa infection, thereby improving our understanding of the "first line of defense" against this bacteria. [unreadable] [unreadable] [unreadable] [unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable][unreadable]